T7 Bacteriophage (Studier, 1972)
In 1972 Studier et al, were studying the T7 bacteriophage. In a search to understand more of T7’s function, Studier et al did a genetic and biochemical analysis to better understand its dynamics as well as interactions with the host cell machinery. The methods used were relatively simple compared to modern methods, being in that the researchers used radiolabeled Carbon based amino acids to perform western blots of proteins produced by the viral genome. The host cell used in their experiments was E.coli. To assure that the proteins being tested were from the phage, the host cells would be treated with high doses of UV light. This UV treatment prevents the host cells from expressing their own genes. In the experiment, mutations causing a functional, both critical and noncritical, change in the bacteriophage, as well as changes in the size and shape of the proteins seen on the western blot. When a mutation occurs in a gene critically denying the ability for the phage to reproduce, this is known as a critical mutation. When a mutation occurs in a gene which isn’t vital for the phage’s reproduction or hinders a protein which can be substituted for a protein already present in the host cell, it is referenced as a noncritical protein. It is not specified in the paper how mutations are elicited within the phage genome, but what is said is that they are ones which either stop transcription part way through the protein, effectively making it shorter, or which prevent the transcription of the gene entirely. In both cases the mutations lead to protein expressions which can be noticeably distinguished on a western blot in comparison to the wild type genome. These changes in the reading of the western blot, in conjunction with differences in efficacy of phage reproduction (or lack thereof), create evidence to deductively reason functionality of proteins under observation. For example, one of the Phage proteins under question was thought to be a DNA ligase protein. So what Studier et al did was they took mutated phage (for the ligase protein) and infected cells with their own ligase and cells void of their own ligase. They found that the mutated Virus could not replicate in the restrictive cells, but could in the permissive cells which had the ligase. They therefore distinguished that this region of the genome, scene as a malformed protein on the western blot, encoded the DNA ligase for the phage. This type of process was similar for the other proteins encoded on T7’s small genome. There are, according to Studier et al, 30 recognizable proteins on a T7’s western blot. These proteins all function in the formation of a new T7 virion. Since the Viral genome is linear, these proteins will be made in succession of one another. Therefore, the RNA polymerase and Ligase are the first two proteins to be encoded on the genome. An interesting note that Studier et al found was that the host polymerase could encode these two, but if the phage RNA polymerase was damaged, then the host Polymerase was drastically unable to encode the rest of the genome after the ligase genes were encoded. This suggests that the viral genome will rely on the host mechanisms in the beginning stages of encoding, but will then have the tools to encode the rest of the necessary virion building blocks. This finding further suggests that mutations in the gene encoding for the phage RNA polymerase are detrimental to the proliferation of the T7 phage. The rest of the paper describes the protein products of the T7 genome and divides them into three classes. The first being comprised of the RNA polymerase and the ligase. The second comprises mainly proteins which breakdown host DNA to synthesize into T7 DNA. The third class has more to do with the proteins creating the capsid and other end product packaging proteins making up the finished virion. References: Studier, F. W. (1972). Bacteriophage T7. Science, 176(4033), 367-376.